Twenty-seven colistin-resistant, carbapenemase-producing Klebsiella pneumoniae isolates were identified from hospitals in Serbia. All isolates were bla CTX-M-15 positive; ST101, ST888, ST437, ST336, and ST307 were bla OXA-48 positive; and ST340 was bla NDM-1 positive. ST307 had an insertion, and ST336 had a premature stop codon in the mgrB gene. Amino acid substitutions were detected in PmrAB of isolates ST101, ST888, ST336, and ST307. The mcr-1 and mcr-2 were not detected. An increase in phoP, phoQ, and pmrK gene transcription was detected for all sequence types.

Klebsiella pneumoniae is a major cause of severe healthcare-associated infections and often shows MDR phenotypes. Carbapenem resistance is frequent, and colistin represents a key molecule to treat infections caused by such isolates. Here we evaluated the antimicrobial resistance (AMR) mechanisms and the genomic epidemiology of clinical K. pneumoniae isolates from Serbia. Consecutive non-replicate K. pneumoniae clinical isolates (n = 2,298) were collected from seven hospitals located in five Serbian cities and tested for carbapenem resistance by disk diffusion. Isolates resistant to at least one carbapenem (n = 426) were further tested for colistin resistance with Etest or Vitek2. Broth microdilution (BMD) was performed to confirm the colistin resistance phenotype, and colistin-resistant isolates (N = 45, 10.6%) were characterized by Vitek2 and whole genome sequencing. Three different clonal groups (CGs) were observed: CG101 (ST101, N = 38), CG258 (ST437, N = 4; ST340, N = 1; ST258, N = 1) and CG17 (ST336, N = 1). mcr genes, encoding for acquired colistin resistance, were not observed, while all the genomes presented mutations previously associated with colistin resistance. In particular, all strains had a mutated MgrB, with MgrB C28S being the prevalent mutation and associated with ST101. Isolates belonging to ST101 harbored the carbapenemase OXA-48, which is generally encoded by an IncL/M plasmid that was no detected in our isolates. MinION sequencing was performed on a representative ST101 strain, and the obtained long reads were assembled together with the Illumina high quality reads to decipher the bla OXA-48 genetic background.

SummaryBackground/Aim: Understanding the composition of bacteria in infected root canals is important for ameliorating the treatment strategies that lead to the elimination of pathogens and infection control, but also prevent reinfection. Aim of this study was to investigate microbial composition of primary infected root canals with apical periodontitis of young permanent teeth, originating form school children in Serbia, and its association with clinical symptoms.Material and Methods: To determine the bacterial composition of infected root canals in children, 35 endodontic samples were obtained. The identification of cultured bacteria was performed by MALDI-TOF MS analysis. The presence or absence of clinical symtoms were recorded.Results: Facultative anaerobes were 2,2 times more frequent than obligate anaerobes. The most common facultative anaerobes belonged to following genera, Streptococcus (58 isolates), Actinomyces (10) and Enterococcus (8), while predominant obligate anaerobes, belonged to genera Veillonella (15), Prevotella (9) and Fusobacterium (8). The most common clinical isolates recovered from infected root canals with symptomatic apical periodontitis were Veillonella parvula (10) and Fusobacterium nucleatum (7), while from the asymptomatic ones, they were Streptococcus mitis/Streptococcus oralis (5). Prevalence of Parvimonas micra, Prevotella buccae and Streptococcus constellatus within the root canals might be associated to clinical symptoms.Conclusions: Species of genera Streptococcus and Veillonella were the most common isolates from primary infected root canals with apical periodontitis in Serbian school children. Facultative anaerobes were predominant over obligate anaerobes. The prevalence of obligate anaerobes was much higher in symptomatic compared to asymptomatic root canal infections. No specific bacterial strain might be associated to a single examined clinical symptom (pain, tenderness to percussion or swelling), but majority of the strains are associated to all of the examined three symptoms.

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Wastewater (WW) is considered a source of antibiotic-resistant bacteria with clinical relevance and may, thus, be important for their dissemination into the environment, especially in countries with poor WW treatment. To obtain an overview of the occurrence and characteristics of carbapenem-resistant Gram-negative bacteria (CR-GNB) in WW of Belgrade, we investigated samples from the four main sewer outlets prior to effluent into international rivers, the Sava and the Danube. Thirty-four CR-GNB isolates were selected for antimicrobial susceptibility testing (AST) and whole-genome sequencing (WGS). AST revealed that all isolates were multidrug-resistant. WGS showed that they belonged to eight different species and 25 different sequence types (STs), seven of which were new. ST101 K. pneumoniae (blaCTX-M-15/blaOXA-48) with novel plasmid p101_srb was the most frequent isolate, detected at nearly all the sampling sites. The most frequent resistance genes to aminoglycosides, quinolones, trimethroprim-sulfamethoxazole, tetracycline and fosfomycin were aac(6′)-Ib-cr (55.9%), oqxA (32.3%), dfrA14 (47.1%), sul1 (52.9%), tet(A) (23.5%) and fosA (50%), respectively. Acquired resistance to colistin via chromosomal-mediated mechanisms was detected in K. pneumoniae (mutations in mgrB and basRS) and P. aeruginosa (mutation in basRS), while a plasmid-mediated mechanism was confirmed in the E. cloacae complex (mcr-9.1 gene). The highest number of virulence genes (>300) was recorded in P. aeruginosa isolates. Further research is needed to systematically track the occurrence and distribution of these bacteria so as to mitigate their threat.